Morphological Observation And Related Gene Analysis Of Metamorphosis Of Capitulum Mitella Larvae

Capitulum mitella belongs to the subclass of Crustacea,Cirripedia,Thoracica,Pollicipidae,Capitulum.It is an important economic species of intertidal zone in China.Because of its unique ecological status,it is also an important species for evolutionary biology and ecological research.In this paper,the morphological changes of the larva during the metamorphosis and development of the C.mitella were described in morphology.Combined with the data of the whole genome and transcriptome of the research group,one HMGR gene,four CaMs genes and seven CthDs genes were identified by the method of bioinformatics.The three gene sequences of CmHMGR,CmCaM1 and CmCthD1 were analyzed by bioinformatics.The expression levels and expression organs of CmHMGR,CmCaM1 and CmCthD1 in different development stages of the larvae were also analyzed by real-time fluorescence quantitative PCR and whole in situ hybridization,providing scientific basis for further understanding the metamorphosis and development process of the larvae.The main results are as follows:1.Observation on the metamorphosis of the larvaThe metamorphosis of the larva include two processes:from nauplius stage to cyprid,and from cyprid to juveniles.In this paper,the evolution of the internal morphology of the larva in the metamorphosis process was observed by means of tissue section,and the changes of the compound eye,single eye,digestive tract,chest limb,antennae,chalk gland,oil cell and other organs were described.2.Identification and analysis of HMGR,CaM and CthD gene familiesThe CmHMGR gene encodes 889 amino acids with a molecular weight of 9.7kda and an isoelectric point of 5.69.It contains one conserved domain of HMG?Co A red and strol sensing,two binding sites of HMG?Co A and two binding sites of NADP(H).The protein sequences encoded by the four CmCaMs genes are composed of 137-224 amino acids with a molecular weight of about 1.5-2.4kda and an isoelectric point of 4.09-5.34,and all of them have two EF-hand7 binding residues and four Ca²⁺binding sites.NJ evolutionary tree shows that the four CmCaMs are clustered into one branch,which is closely related to each other.The protein sequences encoded by the seven CmCthDs genes are composed of 332-397 amino acids,with a molecular weight of about3.5-4.4kda and an isoelectric point of 4.35-9.25.All of them contain Asp domain and two aspartic protease active sites.CmCthD3 contains an A1?propeptide domain.From the NJ tree cluster analysis,we can see that seven CmCthDs are clustered into one branch,which is conservative in evolutionary degree.3.Bioinformatics analysis of CmHMGR gene,CmCaM1 gene and CmCthD1 geneCmHMGR gene is 2667bp,molecular formula is C₄₂₆₁H₆₈₅₃N₁₁₈₅O₁₂₇₃S₅₁,it is hydrophobic protein,it has 68 phosphorylation sites,N-terminal signal peptide without hydrophobic structure,it is transmembrane protein,it has 4 transmembrane regions,?-helix and irregular curl are the largest structural elements in the secondary structure of CmHMGR.The evolution tree showed that CmHMGR was closely related to insects,but not to Homarus americanus and Portunus trituberculatus,CmHMGR was closely related to insects.CmCaM1 gene is 732bp,CmCaM1 protein molecular formula is C₇₁₄H₁₁₂₉N₁₈₇O₂₅₅S₁₁,which is hydrophilic protein,has 13 phosphorylation sites,no transmembrane region and signal peptide,the largest structural element in the secondary structure of?-helix CmCaM1.From the results of the evolutionary tree,CmCaM1 and insects and crustaceans gather into one branch,which shows that CmCaM1 has a conservative evolutionary position in pan crustaceans.It shows that CaM plays an important role in function.CmCthD1 is 1191bp,its molecular formula is C₁₉₀₄H₂₉₄₁N₅₁₅O₅₈₀S₁₅.It is a hydrophilic protein with an N-glycosylation site(Asp193),a signal peptide sites between 1-18 amino acid,no transmembrane region and extended chain and irregular curl are the largest structural elements in the secondary structure of CmCthD1 protein.From the results of the evolutionary tree,CmCthD1 clusters with invertebrates such as crustaceans,insects and molluscs,but forms a branch of its own,indicating that the gene is both conservative and unique in evolution.4.Expression of CmHMGR,CmCaM1 and CmCthD1 genesThe results of fluorescent quantitative PCR showed that the expression of CmHMGR,CmCaM1 and CmCthD1 genes were different in different stages of development of testes:the expression of CmHMGR gene reached the highest level in the?stage of nauplius larva,which promoted nauplius larva to metamorphose to cyprid,and the expression of CmHMGR gene was lower in the third day of nauplius larva,early metamorphosis,middle metamorphosis and immature stage To complete metamorphosis of cyprid to juveniles.The expression level of CmCaM1 gene in cyprid was higher than that in nauplius and juveniles,indicating that CmCaM1 gene may play an important role in metamorphosis of cyprid.The expression level of CmCthD1 gene was high in the stage?nauplius,and very low from the stage?nauplius to the stage of metamorphosis.The expression level of CmCthD1 gene was the highest in the larvae,indicating that CmCthD1 gene played an important role in the growth and development of the larvae.The results of whole in situ hybridization showed that CmHMGR gene could detect positive signals in the head and appendages of stage VI nauplius larva,and expressed strongly in stomach and intestine.From cyprid to juveniles,CmHMGR gene was expressed specifically in the antennae of the head,in the vicinity of the obturator muscle in the chest and abdomen,and in the thoracic nerve nodes.It was speculated that CmHMGR gene might be involved in the metamorphosis and attachment of the larva In addition,the strong positive signals near the adductor indicate that the greater mandibular organ(MO)is likely to be located in the area near the adductor.In the?stage of nauplius,the CmCaM1 gene is located in the head,chest and abdomen positive signals can be detected everywhere in the appendages,indicating that CmCaM1 is widely involved in the regulation of various tissues in the process of metamorphosis from nauplius to cyprid.The expression of CmCaM1 in antennae,cement gland,compound eyes and nauplius eyes,the appendages of cyprid to juveniles is speculated that CmCaM1may be involved in the movement of the thorax,the secretion of adhesive protein and the movement of compound eyes and nauplius eyes.In the?stage of nauplius,CmCthD1gene can be detected very obvious positive signals in the head,thorax,abdomen and appendages.The wide distribution of the tissue indicates that CmCthD1 may be involved in the movement of the thorax,the secretion of adhesive protein and the movement of compound eyes and nauplius eyes.It is important to maintain the homeostasis and normal metabolism of the cells in the body.At the stage from cyprid to juveniles,CmCthD1 gene has positive signals in the antennae and abdomen of the larvae,which indicates that CmCthD1 has an important influence on the metamorphosis of the larva.